1. Field of the Invention
The invention generally relates to apparatus for performing assays, such as chemical assays and biochemical reactions, or the like, at reaction sites on a substrate. In particular, the invention relates to apparatus for performing assays, such as chemical assays and biochemical reactions by delivering a selected aliquot or selected aliquots to a reaction site or sites on a substrate that may include a plurality of layers of semiconductor material.
2. Description of the Related Art
Until the relatively recent advent of combinatorial chemistry and genetic research spawned the need for high-throughput analyzing and screening techniques, researchers performed such assays using vials, tubes, and beakers. However, with ever more substances available via synthesis or via combinatorial techniques for testing, the need has arisen to test the possible role of thousands, or even millions of substances, in comparable numbers of possible reactions. Miniaturization has been identified as a promising path to more efficient, e.g., less expensive, chemical, and in particular, drug, analysis and screening. Discussions of various aspects of such analysis and screening techniques are found in J. D. Devlin, ed., High Throughput Screening: The Discovery of Bioactive Substances (Marcel Dekker, Inc., New York, 1997); which is incorporated herein by reference to more fully describe the state of the art to which the present invention pertains.
Miniaturization apparatus may be broadly classified into at least two categories. A first category involves the placement of chemical substances in small amounts in sites formed on glass or a similar substrate. Micro-chemistry includes processes carried out in small volumes, e.g., between nanoliter and microliter aliquots, whereby reaction times may be shortened significantly over those conducted in reaction vessels holding on the order a fraction of a milliliter, as currently achievable by a lab technician working xe2x80x9cby hand.xe2x80x9d In addition to microchemical testing, levels of gene expression may be tested on a large scale.
An example of this first category is the development of microplate technology in which a glass substrate may include site densities of about 10,000 sites. This technology may include the use of complex micro-robotics or the adaptation of ink-jet technology to apply chemical and biochemical substances to chosen sites on the substrates. Frequently, at least one of the reactants in a chemical assay to be performed is chemically linked to or otherwise immobilized at the reaction site. This is done, so that fluids may be added to and removed from the reaction site without removing at least one intermediate or end product of the reaction, which intermediate or end product(s) is (are) to be retained at the reaction site, so that the outcome of the chemical assay may thereby be detected.
Orchid Biocomputer (xe2x80x9cOrchidxe2x80x9d) of Princeton, N.J., USA, has indicated that it plans to create a credit-card sized glass chip with 10,368 sites. See M. Leach, Update: Discovery on a Credit Card?, DRUG DISCOVERY TODAY, 253-4 (Vol. 2, No. 7 (July 1997). For example, each site may cover an area of 100 xcexcm2 and may contain less than 1 xcexcl in volume. The chip is a glass sandwich formed from individual chip layers, which then may be glued together to form tubes to move substances between sites. Such tubes are formed in this device by cutting, e.g., etching, trenches or grooves in a first layer and then sandwiching the trenches under a second layer.
A second category of miniaturization apparatus employs silicon in some functional, e.g., electrical or mechanical, modality as the substrate, and chemicals then are tested on the substrate. In some cases, micro-robotics or micro-chemistry, or both, may be employed with such substrates. For example, Orchid""s chip may employ microfluidic pumps, e.g., electronic pumps having no moving parts, to move substances between sites. Nanogen, Inc. also has developed a microelectronic device for handling low-dilutions of charged molecules. However, unlike Orchid, which may use electrokinetic pressure pumping, the Nanogen device employs electrophoresis as a motive agent to analyze chemical reactions acting over the surface of the silicon substrate at about twenty-five reaction sites. Electrokinetic pressure pumping has been described as a combination of electrophoresis and electro-osmosis.
Other research has addressed developing products which employ in-place silicon substrates or devices for chemical testing that include either electrical or micro-mechanical technologies, or both. For example, Synteni, Inc. has developed a process which simultaneously measures the expression of thousands of genes using microscopic cDNA portions placed on a substrate. Synteni also has developed a companion reader that uses two-color fluorescence hybridization detection. Genometrix, Inc. also employs a fluorescence analysis technique that appears similar in concept to the Synteni""s process, but carries out the reactions on miniature scale, i.e., on a film that eventually fits over the surface of a reaction reader. Such a reader is manufactured from a silicon chip or wafer modified to function as a photodetector, such as a charge-coupled device (CCD).
Fluorescence generated on the film produces a photocurrent, which provides an electrical charge to a CCD site, and which subsequently may be gated out for analysis, in a manner analogous to the function of a CCD detector array in a digital camera. Thus, known digitizing technology may be combined with the placement of an arrays of chemicals on the surface of a plastic film. The plastic reaction array film may be fitted over the surface of silicon chip or wafer that acts as the reader and when ultra-violet light is flooded over the film surface, fluorescence is elicited from the chemical reaction sites. Each reaction site on the film is aligned with an analyzing site on the reader, and, therefore, a coordinate on the reader corresponds to a reaction site in the chemical array.
Nevertheless, previous attempts to achieve high-throughput analyzing and screening techniques for chemical reactions have required complex operations using combinations of films and substrates or complex robotics for the precise placement of fluids carrying chemical compositions, or both. Such complex systems are subject to failure due to the failure of any system component. Further, such complex systems, especially those including robotics, are expensive to manufacture and maintain.
Thus, a need has arisen for an efficient, simple to operate, and relatively low cost apparatus for performing a high throughput of chemical assays and biological reactions at reaction sites on a substrate.
A further need has arisen for apparatus which allows high-throughput analyzing and screening techniques for chemical assays of biochemical reactions within an aliquot or between aliquots to be performed at discrete reaction sites. Further, it is a feature of the invention that the delivery of aliquot(s) may be accurately and automatically controlled and monitored, e.g., by a rotatable substrate and a movable fluid dispenser. It is a technical advantage of the invention that etch geometry may be used to form reaction sites which may have added advantages in that they may reduce evaporation and aid in the retention of a portion of the fluid.
Yet a further need has arisen for easily assembled and simply and accurately controlled apparatus for delivering an aliquot or aliquots to reaction sites in order to perform chemical or biochemical testing, or both. It is a feature of the invention that the apparatus achieves a high degree of accuracy in the delivery of fluids to reaction sites. It is a technical advantage of the apparatus that it may employ prepackaged engines or motors, such as linear and rotary stepper motors, to move and position at least one fluid dispenser outlet over a reaction site. Such stepper motors provide a high degree of accuracy and repeatability of movement. Such stepper motors also permit the use of an integrated control system with electronic damping and an integrated indexing system. Moreover, the control systems for such stepper motors may readily be customized to provide for variable speed and continuous speed operation.
Still a further need has arisen for an apparatus which aligns at least one fluid dispenser outlet with at least on reaction site without the use of complex robotics. It is a technical advantage that the linear stepper motor(s) move(s) the fluid dispenser outlet(s) in one dimension along at least one rail, and that the rotary stepper motor(s) rotate(s) the substrate around an axis. It is a further technical advantage of the use of linear and rotary stepper motors that they may be less expensive to manufacture, maintain, and replace than complex robotics.
Yet another need has arisen for an apparatus having a multi-function head comprising at least one fluid dispenser for delivering a fluid or fluids to one and at least one of a plurality of reaction sites and at least one readout device.
The readout device(s) may serve a plurality of functions including monitoring the progress of assays, scanning the reaction site(s) to determine the results of assays, locating a reaction site or sites by reading a locating mark, and guiding at least one dispenser outlet to a reaction site or sites by means of a tracking mark. It is a technical advantage of the multi-function head that the operation and construction of the apparatus is simplified by the combination of multiple functions on a single movable head. It is a further technical advantage of the multi-function head that a single control system may position both the at least one fluid dispenser and the at least one readout device, thereby eliminating alignment differences between these components. It is yet another technical advantage of the multi-function head that rapidly or instantaneously occurring assays may be monitored immediately after initiation and monitored until completion. It is still another technical advantage of the apparatus that a micropositioner, such as a three-axis micropositioner, may be controlled to make adjustments, e.g., adjustment is a range of less than about 15 mm with an accuracy of about one micron, along Cartesian axes in the position of the at least one fluid dispenser outlet and the readout device.
The invention is an apparatus for performing a plurality of assays, such as a plurality of chemical assays or a plurality of biochemical reactions comprising an axially rotatable substrate including a plurality of radially-arrayed reaction sites. Other assays include cellular assays as well as physical and biophysical assays, e.g., chemiluminescence luminescence, dielectric field strength, resistivity, impedance, circular dichroism, refractivity, surface plasmon resonance, optical absorbance, magnetic resonance, and the like. Assay components may include, for example, synthetic organic compounds (e.g., compounds of less than 100,000 molecular weight, preferably compounds of less than 10,000 molecular weight, more preferably compounds of less than 1,000 molecular weight) proteins (e.g., enzymes, amyloid proteins, receptors, cytokines, and antibodies) peptides, oligopeptides, nucleic acids (including modified synthetic derivatives thereof, DNA, RNA oligonucleotide and monomeric nucleotides, nucleosides, modified synthetic variants thereof, and the like) cells (e.g., bacterial cells; yeast or other fungal cells; unicellular organisms such as protozoans; animal cells including insect, avian, and mammalian cells; and plant cells) cell membranes and other cellular components, buffers, salts, ions such as metal ions, lipids, carbohydrates, vitamins, extracellular matrixes or components thereof, as well as blood serum, or other bodily fluids.
The substrate may be manufactured from glass, ceramics, semiconductor materials, plastics, composites, and combinations thereof. Semiconductor materials are solid crystalline materials whose electrical conductivity is intermediate between that of a conductor and an insulator, ranging from 105 mhos and 10xe2x88x927 mho per meter and is usually strongly temperature dependant. Semiconductor materials may include silicon, germanium, and gray tin. For example, the substrate may include a plurality of layers of semiconductor material which may partially or completely cover the surface of the substrate. Alternatively, the plurality of layers may lie beneath the surface of the substrate and extend for a portion or for the entire area of the substrate.
The apparatus further comprises means for rotating and controlling the rotation of the substrate and at least one fluid dispenser for conveying at least one fluid to at least one of the reaction sites. The means for rotating may comprise a engine, such as an air driven turbine, or a motor. Each of such fluid dispensers includes a fluid dispenser outlet. In addition, the apparatus includes means for identifying the at least one reaction site, and means for aligning the at least one fluid dispenser outlet with the at least one reaction site.
In particular, the apparatus may comprise at least one multi-function head, such as a dual function head, including at least one fluid dispenser for conveying at least one fluid to at least one of the reaction sites and at least one readout device. The readout device may include means for locating a reaction site, such as the means for identifying a location mark, and for monitoring the chemical or biochemical reactions at the reaction sites. Each of such fluid dispensers includes a fluid dispenser outlet. Thus, the fluidics and locating and monitoring functions of the apparatus may be combined in a multi-function head.
The operation of stepper motors is known in the art. For example, such motors are used in computer disk drives. Generally, a stepper motor rotates in short, essentially uniform regular movements. The stepped movements are obtained by means of electromagnetic controls. Although the apparatus may include a rotary stepper motor, the means for rotating also may rotate the substrate at an adjustable or substantially continuous speed, or both, and may control the rotation of the substrate by adjusting the speed and a direction of rotation. Further, the means for rotating is controllable to rotate the substrate at a speed, such that a portion of the at least one fluid is removable from the at least one reaction site by a centrifugal force generated by the rotation of the substrate. Moreover, at least one channel may join the at least one reaction site to at least one other reaction site. The means for rotating further may be controllable to rotate the substrate at a speed, such that the at least one fluid is drawn from the at least one reaction site through the at least one channel to the at least one other reaction site by a centrifugal force generated by the rotation of the substrate.
The fluid(s) delivered to the reaction site may comprise at least a first amount of at least one fluid aliquot and at least a second amount of at least one separating fluid, e.g., a solvent, oil, air, immiscible fluid, or the like. For example, the first amount of at least one fluid aliquot may be substantially identical to the second amount of at least on separating fluid. In another embodiment, however, the first amounts of the at least one fluid aliquot may be substantially identical to each other while the second amounts of the at least one separately fluid are of a different amount and are substantially identical to each other. For example, an oil or air may be a preferred separating fluid for water-based aliquots. Further, the at least one fluid dispenser may include one or more pumps, suction devices, and timing devices for controlling the pump(s) or the suction device(s), or both. The pump(s) may include conduits and valves, whereby the pump(s) may alternately draw at least one of the first amount, e.g., in a range of about 0.0001 to 5 xcexcl, and preferable about 3 to 5 xcexcl, of the at least one fluid aliquot and at least one of the second amount of the at least one separating fluid into the dispenser tube and delivers an alternating stream of the at least one aliquot and the at least one separating fluid to the at least one fluid dispenser outlet under a controlled pressure differential relative to the ambient pressure surrounding the fluid dispenser outlet(s). The timing device(s) then may control the operation of the suction device(s), such that the suction device(s) may draw off the stream from the fluid dispenser outlet(s).
Specifically, the timing device(s) may measure a flow rate of the stream through the dispenser tube and deactivate and subsequently reactivate the suction device(s), such that at least one first amount of the at least one aliquot is delivered to the reaction site. The suction device(s) may create a suction pressure less than the ambient pressure surrounding the dispenser outlet(s), e.g., a vacuum sufficient to remove fluid from the dispenser outlet(s). Alternatively, a plurality of suction devices may create different degrees of pressure differential across the orifices of such suction devices, e g., different levels of vacuum, with respect to the ambient pressure surrounding the dispenser outlet(s). In still another alternative, a library of tubes may be provided, each tube having a predetermined amount of a chemical or solution for use in performing a chemical assay or causing biological reaction. A desired amount of the chemical solution may then be drawn or pumped from the tube and deposited at a reaction site or reaction sites. The unused portion of the chemical or solution may be discarded or recovered for recycling or reuse, or the tube also may be discarded or refilled, sealed, and reused. Other means for dispensing or removing fluids at reaction sites also may be used in accordance with the invention. See, e.g., D. W. Brandt, Multiplexed Nanoliter Transfers for High Throughput Drug Screening Using the BIOMEK 2000 and the High Density Replicating Tool, J. BIOMOLECULAR SCREENING 2:111-116 (1997); which is incorporated herein by reference to more fully describe the state of the art to which the present invention pertains.
The dispenser outlet(s) may be movably mounted on a rail which transects the substrate and is oriented substantially parallel to a surface of the substrate, e.g., is suspended over the substrate, and a first motor may be used to rotate the substrate. The means for aligning comprises a second motor for positioning the at least one fluid dispenser outlet along the rail. Moreover, as noted above, the first motor may be a rotary stepper motor, and the second motor may be a linear stepper motor. In addition, the means for aligning may comprise a computer (including a microprocessor or other electronic device) which receives, processes, and presents data, and which stores a start location on the substrate""s surface for the dispenser outlet. The computer and additional, functionally linked electronics including, for example, a signal generator such as an electromagnetic energy source, and a calibrating sensor, such as an electromagnetic energy sensor, may provide movement signals to the first and second motor. Thus, the computer and the additional electronics generate signals to align the dispenser outlet over the reaction site. Alternatively, the fluid dispenser outlet(s) may be mounted on a pivotable arm which may be rotated through an arc across the surface of, e.g., over, the rotating substrate. In this embodiment, the second motor may also be a rotary stepper motor.
In addition, the apparatus may position the multi-function head by means of a two step process. First, the apparatus may direct the head to the general vicinity of a selected reaction site. Second, the multi-function head may utilize the means for identifying to interrogate or read the locating marks to identify the selected reaction site and to align the dispenser with that reaction site.
The means for identifying may include at least one sensor. This sensor may be positioned in the same manner as the fluid dispenser outlet, e.g., it may be joined to a linear stepper motor which is mounted on a rail above the substrate. Preferably, the sensor is incorporated into the head. This at least one sensor may receive a signal emanating from the substrate, or the at least one sensor may transmit an interrogating signal and receive a locating signal in response. Further, the at least one sensor may read at least one locating mark, e.g., an indexing mark, a tracking mark, a bar code, or combinations thereof, on the substrate""s surface. Examples of the locating mark are discussed below. See FIG. 7. However, as noted above the locating mark may consist of an indexing mark, which identifies the particular reaction site, and at least one tracking mark which helps the means for aligning to guide the multi-function head and its associated fluid dispenser(s) and readout device(s) over the reaction site. In particular, the tracking mark may be recognized and help guide the head to the reaction site by its size or shape or by its physical relationship to, i.e., distance from or direction to the reaction site.
In another embodiment of the invention, the apparatus for performing a plurality of assays again comprises an axially rotatable substrate including a plurality of radially, arrayed reaction sites; means for rotating the substrate; and at least one multi-function head including at least one fluid dispenser for conveying at least one fluid to at least one of the reaction sites and at least one readout device. The at least one fluid dispenser also may include at least one fluid dispenser outlet. The apparatus also may include means for identifying the at least one reaction site and means for aligning the at least one multi-function head, such that the at least one fluid dispenser outlet is aligned with the at least one reaction site. The means for rotating may be controllable to rotate the substrate at a speed, such that a portion of the at least one fluid is removable from the at least one reaction site by a centrifugal force generated by the rotation of the substrate.
In still another embodiment of the invention, the apparatus for performing a plurality of assays comprises an axially rotatable substrate including a plurality of radially-arrayed reaction sites; means for rotating the substrate; at least one fluid dispenser for conveying at least one fluid to at least one dispersion point, preferably located on the substrate; and means for identifying at least one of the reaction sites. Further, the apparatus, and preferably the substrate, may include at least one channel joining the at least one dispersion point to the at least one reaction site. Alternatively, this embodiment may include at least one multi-function head including at least one fluid dispenser for conveying at least one fluid to at least one dispersion point and at least one readout device. The means for rotating may be controllable to rotate said substrate at a speed, such that the at least one fluid is conveyed from the at least one dispersion point to the at least one reaction site by a centrifugal force generated by the rotation of the substrate.
In yet another embodiment of the invention, the apparatus for performing a plurality of assays comprises an axially rotatable substrate including a plurality of radially-arrayed reaction sites; at least one fluid dispenser for conveying at least one fluid to the substrate; means for identifying at least one of the reaction sites; and means for rotating the substrate. For example, the at least one fluid dispenser may convey at least one fluid to the substrate through a spindle around which the substrate rotates. The means for rotating is controllable to rotate the substrate at a speed, such that the at least one fluid is drawn across the reaction sites by a centrifugal force generated by the rotation of the substrate.
Other features and technical advantages will be apparent to persons skilled in the relevant art in view of the following detailed description and accompanying drawings.